The goal of this research is to determine whether the virulence of Candida albicans can be reduced by changing the structure of the cell wall, and if so, to exploit this information for the development of new anti-fungal drugs. The cell wall, because it is involved in attaching in fungus to host cells and in protecting the fungus from host immune cells, may play a significant role in pathogenicity. We have isolated several C. albicans mutants, designated pcd, that have a 5- to 10-fold reduction in chitin, a cell wall polysaccharide, and have shown that they are significantly less virulent than the wild-type parent. To establish unequivocally that the loss of chitin and the decrease in pathogenicity are both due to the same mutation, we will clone and disrupt the PCD gene(s). We will also construct a mutant defective specifically in chitin synthesis by disrupting the C. albicans homolog of Saccharomyces cerevisiae CSD2. In S. cerevisiae, mutations in CSD2 cause a 10-fold reduction in cell wall chitin and a loss of chitin synthase III activity. Characterization of the C. albicans disruptant will include analyses of cell wall chitin composition, chitin synthase activity, virulence in mice, and adherence to host cells. We will develop high capacity screens to detect inhibitors of the PCD and/or CSD genes or gene products. To look for inhibitors of chitin synthesis, we will pulse-label C. albicans glycocyamine deaminase mutants with [3H]-glycocyamine and monitor the synthesis of radioactive, alkali- insoluble material. To look for inhibitors of the expression of a PCD gene, we will fuse the Candida kefyr LACZ gene to the promoter of the PCD gene and assay beta galactosidase activity. Our approach is novel in two respects. First, our targets will be cellular components that are essential for growth. Second, our screen for inhibitors of chitin synthesis will detect chitin synthase III rather than the trypsin-stimulated chitin syntheses. Fungal cells contain multiple chitin syntheses, and studies in S. cerevisiae indicate that most of the chitin in the cell is made by chitin synthase III, a trypsin- independent enzyme that requires three genes, CSD2, CSD4, and CAL3, for activity. Thus, chitin synthase III potentially provides three new targets for the development of anti-fungal agents.